PROJECT SUMMARY ? PROJECT 1 We aim to cure sickle cell disease (SCD) by merging new technologies for gene manipulation with recent insights into the perinatal ?-to-? globin gene switch. SCD is caused by mutations in HBB, which encodes the ?-globin subunit of adult hemoglobin (HbA, ?2?2). Elevated fetal Hb (HbF, ?2?2) caused by persistent postnatal??-globin (HBG1 and HBG2) gene expression alleviates pathologies of SCD. In hereditary persistence of fetal hemoglobin (HPFH), HbF exceeds 20% in all adult red blood cells (RBCs) and co-inherited SCD is clinically silent. Modern genetic studies reveal that the ?-to-? globin switch is mediated by BCL11A, a transcription factor that binds cis elements in the extended ?-globin locus, where contiguous HBG2, HBG1 and HBB genes compete for an upstream enhancer, termed locus control region (LCR). Hence, manipulation of human hematopoietic stem cells (HSCs) to reduce erythroid BCL11A expression or ablate its binding sites in the extended ?-globin locus favors HBG1/HBG2-LCR interactions and HbF expression in erythroid progeny. We will study both approaches as potential new gene therapies for SCD. Aim 1 is to develop novel lentiviral vectors (LVs) that express erythroid- specific BCL11A shRNA. Studies by others show that transduction of CD34+ cells with an LV encoding BCL11A shRNA driven by erythroid-specific regulatory elements raises HbF in RBCs generated by in vitro differentiation. However, this LV exhibits relatively poor hematopoietic stem cell (HSC) transduction, as measured by vector copy number (VCN) after long-term reconstitution of immunodeficient mice. We built two novel LVs that increase HSC transduction efficiency by 5- to 8-fold and raise RBC HbF to potentially therapeutic levels. We will study our novel LVs using in vitro culture assays and animal models to acquire additional preclinical efficacy and safety data and develop a production process (with GMP Core C) to support a clinical trial for adult SCD patients by year 3 of this study. Aim 2 utilizes genome editing-mediated non-homologous end joining (NHEJ) to raise adult RBC HbF, either by disrupting an erythroid-specific BCL11A gene enhancer, or by recapitulating a benign, naturally occurring form of HPFH caused by a 13-nucleotide HBG1 promoter deletion. In preliminary studies, both approaches raised HbF to potentially therapeutic levels in RBCs derived from normal or SCD patient CD34+ cells. We will optimize and compare these two gene editing approaches in cultured CD34+ cells, animal models and in vitro assays to identify the optimal method for altering HSCs to induce RBC HbF therapeutically with minimal off-target genotoxicity. Current gene therapy for SCD is promising, but expensive and not consistently effective. By comparing several new LV and gene editing approaches simultaneously, we hope to identify the safest, most effective and economical approach for curing this devastating disease that affects hundreds of thousands of Americans and millions of individuals worldwide.